Introduction
Chitobiase, from Vibrio harveyi, is a membrane bound lipoprotein involved in the degradation of chitin. Chitobiase is similar to and may share a common ancestry to the a-chain of human b-hexos-aminidase. Chitobiase is encoded by chb.
In this experiment, a restriction map for restriction enzymes Eco R1, Pst1 and Hind III using Southern hybridization and restriction analysis of pRSG 192. pRSG 192 is a recombinant plasmid derived from the chb gene and pUC 19, a 2.7kb engineered plasmid which encodes for ampicillin resistance, a portion of the lac operon and a multiple cloning region . The chb gene exists as a 3.6 kb insert in the mutiple cloning region of pUC 19.
The major goals of Experiment One will be to isolate pRSG 192 from an overnight culture of E. coli, amplify a region of the chb gene using PCR, and to map restriction sites within the chb gene using restriction analysis and Southern hybridization.
Methods
Plasmid Isolation
Four microfuge tubes containing cell pellets representing 3.0ml of cells(2 x 1.5ml) from an overnight culture of E. coli were prepared. The supernatant fluid was discarded and each pellet was resuspended in 150ul of TE buffer(10mM Tris-HCl, pH 8.0; 0.1 EDTA). 300ul of SDS(1% SDS, 0.2 N NaOH) was added to each pellet. The tubes were placed on ice for five minutes, after which, 225ul of ice-cold 3M potassium acetate(pH 4.8) was added. The tubes were again placed on ice for five minutes and subsequently microfuged for five minutes.
The supernatants were recovered and transferred to new tubes. One volume of phenol/chloroform was added to each new tube. The tubes were shaken vigorously for two minutes and centrifuged for five minutes. The upper, aqueous phase was recovered and transferred to a new tube. One volume of chloroform was added to each tube. The tubes were vigorously mixed and microfuged for three minutes.
The miniprep consisted of isolating the DNA plasmid from the bacterial cells. This was used to identify the success of EGFP ligation into pET41a(+) vector upon restriction digest and gel electrophoresis. Additionally, Polymerase Chain Reaction (PCR) was run on the isolated DNA plasmids with one of the primers specifically annealing to a part of pET41a(+) sequence and the other annealing to the EGFP gene.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
20.0cm3 of 0.10M ethanoic acid was pipetted into a conical flask. 3. 0.10M sodium hydroxide solution was titrated using phenolphthalein as indicator, until the solution was just turned pink. 4. A further 20.0cm3 of the same ethanoic acid solution was added to the flask and was mixed thoroughly.
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
It has an outer membrane that contains lipopolysaccharides, a periplasmic space with a peptidoglycan layer, and an inner cytoplasmic membrane. It also consists of adhesive fimbriae. Some strains of E. coli are piliated and are capable of accepting, as well as transferring plasmid to and from other bacteria. This enables the bacteria under stressful or bad conditions to survive. Although its structure is simple with only one chromosomal DNA and a plasmid, it can perform complicated metabolism to help maintain its cell division and cell growth. E. coli produce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight (phschool.com). It is the preferred bacteria in most laboratories because it grows fast and easy, and can obtain energy from a wide variety of sources. Since the birth of molecular cloning, E. coli has been used as a host for introduced DNA sequences (biotechlearn.org.nz). In 1973, Boyer and Cohen showed that two short pieces of DNA could be cut and pasted together, and returned to
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
Talaro , K., & Chess, B. (2012). Foundations in microbiology. (8th ed., pp. 563-564). New York, NY:
...Brighter appearance to coloured textiles thanks to a new cellulase from an extremophilic bacterium. Journal of Biotechnology 66, 231–233.
Once the mixture had been completely dissolved, the solution was transferred to a separatory funnel. The solution was then extracted twice using 5.0 mL of 1 M
Caplan M. Geralyn. DNA Isolation Lab. Owensboro Community & Technical College, n.d. Web. 5 April 2014.
In this method, living spores which are resistant to whichever sterilizing agent is being tested are prepared in either a self contained system, such as dry sp...
2. In the large beaker, put water and boil it completely. After that, remove the beaker from heat. 3. Sample tubes (A-D) should be labeled and capped tightly.